The mitochondrion is now emerging as a site in vascular cells capable of regulating redox cell signaling events in the cytosol. The mechanisms through which this occurs are not well understood and are the focus of this proposal. Our recent studies suggest that the induction of the endogenous intracellular antioxidants through the Keap1/Nrf-2 system is modulated by post-translational modification of mitochondrial protein thiols. This is important because it is known that deficiencies of endogenous antioxidant proteins, such as heme oxygenase-1, in the vasculature are associated with increased development of atherosclerotic lesions, while over-expression is protective. Interestingly, we have shown that modification of mitochondrial protein thiols leads to endothelial cell dysfunction by inhibition of the endogenous antioxidant pathway. In addition, mitochondrial thiol modification results in the attenuation of the endothelium-dependent vasorelaxation of aortic rings in response to acetylcholine. Our preliminary data also demonstrate that mitochondrial thiol modification causes a switch in bioenergetic pathways away from mitochondrial oxidative phosphorylation and toward the less efficient glycolytic pathway, potentially contributing to a bioenergetic deficiency. Taken together these findings have led to the hypothesis that modulation of mitochondrial protein thiol and redox status will alter vascular endothelial cell function. This will be tested by pursuit of the following specific aims: 1: Determine the role of mitochondrial protein thiols and redox status in the susceptibility of endothelial cells (EC) to oxidative stress; 2: Determine the role of mitochondrial thiol modification and altered redox status in vascular endothelial dysfunction; and 3: Determine the mechanistic role of mitochondrial protein thiol modification in endothelial cell dysfunction during increased mitochondrial oxidative stress in atherosclerotic mouse models. The information gained from the accomplishment of these specific aims will give insight into the role of the mitochondrion, particularly mitochondrial protein thiols, in determining endothelial cell dysfunction associated with atherosclerosis.